- Volume 97, Issue 3, 2016

Recombination in alphaherpesviruses allows evolution to occur in viruses that have an otherwise stable DNA genome with a low rate of nucleotide substitution. High-throughput sequencing of complete viral genomes has recently allowed natural (field) recombination to be studied in a number of different alphaherpesviruses, however, such studies have not been applied to equine herpesvirus 1 (EHV-1) or equine herpesvirus 4 (EHV-4). These two equine alphaherpesviruses are genetically similar, but differ in their pathogenesis and epidemiology. Both cause economically significant disease in horse populations worldwide. This study used high-throughput sequencing to determine the full genome sequences of EHV-1 and EHV-4 isolates (11 and 14 isolates, respectively) from Australian or New Zealand horses. These sequences were then analysed and examined for evidence of recombination. Evidence of widespread recombination was detected in the genomes of the EHV-4 isolates. Only one potential recombination event was detected in the genomes of the EHV-1 isolates, even when the genomes from an additional 11 international EHV-1 isolates were analysed. The results from this study reveal another fundamental difference between the biology of EHV-1 and EHV-4. The results may also be used to help inform the future safe use of attenuated equine herpesvirus vaccines.

In this study, we have deleted the lef8 gene of the baculovirus BmNPV, which encodes one of the viral RNA polymerase subunits, in order to create a knockout bacmid, Δlef8, directing cytopathology-free single-cell infections for gene transduction and recombinant protein production. However, while removal of the complete lef8 ORF produced the expected phenotype, it also affected the function of the closely linked essential gene orf40, thus hampering the mutant bacmid rescue in cultured Bombyx cells expressing recombinant LEF8. Subsequently, we determined that several diverse sequences can substitute for the orf40 5′-upstream sequences that were removed by the deletion of the lef8 gene and also showed that neither a physical linkage nor expression of the two relevant genes under native promoter control is a prerequisite for a fully functional virus. Based on these findings, we generated a rescue-competent lef8-null vector, which contained a heterologous promoter-driven orf40. This lef8-deficient vector, which produces productive infections and progeny virus lacking lef8 in deficiency-complementing cells expressing LEF8, could be used as the basis for an alternative to current silkmoth transduction systems.

In the past decade, the incidence and distribution of the recombinant, tuber necrotic strain of Potato virus Y (PVYNTN) has been increasing in the US seed potato crop while the ordinary strain (PVYO) has been decreasing. The transmission efficiency of both strains was determined from two potato cultivars when acquired sequentially by the same aphid or when acquired by separate aphids and inoculated to the same plant. PVYNTN was transmitted more efficiently than PVYO and the order of acquisition or inoculation did not affect the preferential transmission of PVYNTN. When a recipient plant became infected with both strains, PVYNTN maintained higher titre than PVYO and would facilitate the acquisition of PVYNTN. Furthermore, the acquisition and transmission of PVYNTN over PVYO was enhanced in the potato cultivar that expressed a strain-specific Ny-like resistance gene that confers partial resistance to PVYO.

Real-time quaking-induced conversion (RT-QuIC) is a rapid, specific and highly sensitive prion seeding activity detection assay that uses recombinant prion protein (rPrPSen) to detect subinfectious levels of the abnormal isoforms of the prion protein (PrPSc). Although RT-QuIC has been successfully used to detect PrPSc in various tissues from humans and animals, including sheep, tissues from goats infected with classical scrapie have not yet been tested. Therefore, the aims of the present study were to (1) evaluate whether prion seeding activity could be detected in the brain tissues of goats with scrapie using RT-QuIC, (2) optimize reaction conditions to improve scrapie detection in goats, and (3) compare the performance of RT-QuIC for the detection of PrPSc with the more commonly used ELISA and Western blot assays. We further optimized RT-QuIC conditions for sensitive and specific detection of goat scrapie seeding activity in brain tissue from clinical animals. When used with 200 mM sodium chloride, both full-length sheep rPrPSen substrates (PrP genotypes A136R154Q171 and V136R154Q171) provided good discrimination between scrapie-infected and normal goat brain samples at 10− 3 dilution within 15 h. Our findings indicate that RT-QuIC was at least 10 000-fold more sensitive than ELISA and Western blot assays for the detection of scrapie seeding activity in goat brain samples. In addition to PRNP WT samples, positive RT-QuIC reactions were also observed with three PRNP polymorphic goat brain samples (G/S127, I/M142 and H/R143) tested. Taken together, these findings demonstrate that RT-QuIC sensitively detects prion seeding activity in classical scrapie-infected goat brain samples.